N-Substituted derivatives of bleomycins

ABSTRACT

N-Substituted derivatives of bleomycins, which are useful antibiotic substances having an extremely high anti-microbial activity and an antitumer activity with low toxicity, are easily produced by reacting 3-aminopropylaminobleomycin, 3-(N-3aminopropyl)aminopropylaminobleomycin, 3-(N-4aminobutyl)aminopropylaminobleomycin, 3-(N-6aminohexyl)aminopropylaminobleomycin or 3-(N-methyl-N-3aminopropyl)aminopropylaminobleomycin with a compound of the formula,

United Umezawa et al.

[ N-SUBSTITUTED DERIVATIVES OF BLEOMYCINS [75] lnventors: .Hamao Umezawa, Tokyo; Yuya Nakayama, Yono; Mamoru Kunishima; Midori Matsubara, both of Tokyo, all of Japan [73] Assignee: Nippon Kayaku Kabushiki Kaisha,

Tokyo, Japan 22 Filed: Jan. 7, 1974 21 Appl. No.: 431,130

Related U.S. Application Data [63] Continuation-impart of Ser. No. 291,178, Sept. 22, '1972, abandoned, which is a continuation-in-part of Ser. No. 248,631, April 28, 1972, abandoned.

[30] I Foreign Application Priority Data OTHER PUBLICATIONS J. D. Roberts and M. C. Caserio, Basic Principles of Organic Chemistry, Benjamin, inc. New York, 1965, pp. 563 564, 686.

[ Nov. 25, 1975 Ishizuka et al., J. of Antibiotics, Ser. A, 20, 15-24, (1967).

Primary ExaminerLewis Gotts Assistant ExaminerReginald J. Suyat Attorney, Agent, or FirmSchuyler, Birch, Swindler, McKie & Beckett [57] ABSTRACT N-Substituted derivatives of bleomycins, which are useful antibiotic substances having an extremely high anti-microbial activity and an antitumer activity with low toxicity, are easily produced by reacting 3-aminopropylaminobleomycin, 3-(N-3-aminopropyl)aminopropylaminobleomycin, 3-(N-4-aminobutyl )aminopropylaminobleomycin, 3-( N-6-aminohexyl)aminopropylaminobleomycin or 3-( N-methyl-N-3- aminopropyl)aminopropylaminobleomycin with a compound of the formula,

wherein R is hydrogen (except where Y represents -X or -SO X), a saturated or unsaturated, straight chain or branched chain alkyl, cycloalkyl, phenyl, naphthyl, aralkyl or heterocyclic group, these groups being optionally substituted; and Y is -COOl-l or a reactive derivative thereof, -X (wherein X is a halogen), --SO,X (X is as defined above), -NCO,

(wherein R is an alkyl).

24 Claims, '7 Drawing Figures U.S. Patent Nov.25, 1975 Sheet 1 of7 3,922,262

00m. 00mm 00mm U.S. Patent Nov. 25, 1975 Sheet 3 5f? 3,922,262

.15. mwmzzz w 8% com com 89 0o: oo- 8: 8 08. eoom ooqn woow m QE N-SUBSTITUTED DERIVATIVES OF BLEOMYCINS R, where R is an C,-C, alkyl group, an ally] group, a benzyl group or a p-nitrobenzyl group;

CROSS REFERENCE TO THE RELATED SO2R3, where R3 is an crcz alkyl group,

APPLICATION This application is a Continuation-in-Part of US. Ser. No. 291178 filed on Sept. 22, 1972, which is a Continuation-in-Part of US. Ser. No. 248631, filed on Apr. 28, 1972, both now abandoned.

The present invention relates to novel N-substituted 10 (wherein Z is hydrogen, a methyl group, a chlorine derivatives of bleomycins and a method for preparing atom or a nitro group) or a naphthyl group; the same. More particularly, it relates to N-substituted CONHR where R is an C -C alkyl group, a cyderivatives of bleomycins having the following strucclohexyl group, a phenyl group, a p-tolyl group, or a ture in their acid-containing form, naphthyl group;

if mg 11 0 /NCH2/ (5 I !l I 11 I l a N\ 0 ant o O/CH\CH5CH2\CH2 I CH \N/ CH5 H i H (wherein Z is hydrogen, a methyl group, a nitro group or a methoxy group),

60 (wherein Z is hydrogen, a chlorine atom or a nitro group), a styryl group or a naphthylmethyl group;

where R is hydrogen or a methyl group;

CH2 wherein R is hydrogen or a nitro group;

3 4 -(CH CEN; A is an inorganic anion such as a 6 CO\ chlorine atom or A sulfate radical and x is an integer of Q l to 3, and a method for preparing N-substituted derivatives of bleomycins which comprises reacting 3- aminopropylaminobleomycin, 3-(N-3-aminopropyl- )aminopropylaminobleomycin, 3-(N-4-aminobutyl- )aminopropylaminobleomycin, 3-(N-6-aminohexyl- )aminopropylaminobleomycin or 3-(N-methyl-N-3- aminopropyl)aminopropylzfninobleomycin with one member selected from the group consisting of compounds represented by the formula.

wherein R is as defined above and Y is COOH, an acid halide group, an acid ester group, CO-O- COR, or CON the formula,

wherein n is an integer of 3 to 7; the formula,

R'--CHCO\ CH2CO wherein R is as defined above; the formula,

RC-CO 0 CHCO wherein R is as defined above; the formula,

CH,=CCO

CH,CO

the formula,

wherein R is as defined above; the formula, R X, wherein R is as defined above and X is a halogen atom; the formula, R 0 8 X,

wherein R and X are as defined above; the formula, R-NCO, R is as defined above; the formula,

wherein R is as defined above and R is C -C alkyl group; the formula,

wherein R is as defined above and the formula, CH =CHCN.

N-Substituted derivatives of bleomycins of the present invention usually contain one metal atom, e.g. copper, nickel, iron manganese and the like, in a chelate form, and have one basic group in their main nuclear part and two or less basic groups in their side chain (that is, a side chain PQ has basic groups of two or less). Therefore, N-substituted derivatives of bleomycins are recovered as a salt of inorganic acid, for example, those bonded with 1-3 equivalents of inorganic acid, such as hydrochloric acid or sulfuric acid. pH of an aqueous solution of the aforesaid N-substituted derivatives of bleomycins in an acid-free form, i.e. free base, is 9 0r more, so that said aqueous solution absorbs carbon dioxide gas existing in air and then is converted into carbonate thereof. Thus, isolation of said bleomycins in a pure basic form is relatively difficult.

Anti-microbial activity and carcinostatic activity of N-substituted derivatives of bleomycins of the present invention is displayed by the part represented by the above-mentioned formula. Therefore, N-substituted derivatives of bleomycins are represented by the above-mentioned formula in the present invention. In the present invention, the terms "bleomycins" and N- substituted derivatives of bleomycins" include all forms of bleomycins, i.e. metal-containing", metal-free", acid-containing" or acid-free" forms.

3-Aminopropylaminobleomycin (I) used in the present invention as a starting material is a bleomycin the terminal chain of which is 1,3-diaminopropane. 3-(N- 4-Aminobutyl)aminopropylaminobleomycin (II) is a bleomycin the terminal chain of which is 3-(N-4- aminobutyl)aminopropylamine (spermidine) and is known as bleomycin A 3-N-Methyl-N-3-aminopropyl)-propylaminobleomycin (III) is a bleomycin having an N,N-bis(3-aminopropyl)methylamino group as terminal chain. 3-(N-3-Aminopropyl)aminopropylaminobleomycin (IV) is a bleomycin having a N,N-bis(3-aminopropyl)amine as a terminal chain. 3- (N-6-Aminohexyl)aminopropylaminobleomycin (V) is a bleomycin having a 3-(N-6-aminohexyl)-aminopropylamine. These bleomycins as a metal-free and acid-free form have the following structure:

(I) (ll) a starting material for the synthesis of various valuable NH(CHZ):N(CHz)a NH2 (m) bleomycin derivatives having an improved antitumor C activity, a lower toxicity and a selective antitumor ac- H 2 a z).1NH (w) 5 tivities against tumors of various internal organs. NH(H1)=NH(CH2)6 NH2 (v) As a result of diversified studies on bleomycins. the

present inventors have now found that terminal primary amino group of the bleomycins is very reactive These bleomycins are representative biosynthetic and thereby the bleomycins can be converted into usebleomycins which can be easily produced in a high 10 ful novel carcinostatic derivatives thereof with various yield by adding an amine corresponding to the respecreagents. tive terminal chain amine moiety to a medium in which Based on the said finding, the present invention has bleomycinproducing strains are cultured to prepare been accomplished. bleomycin. The representative modes of reaction for the prepa- The bleomycins themselves have an anti-microbial l5 ration of N-substituted derivatives of bleomycins acactivity and an antitumor activity and are also useful as cording to the present invention are as follows:

Bleomycin Reagent Product la) BLM-NH R-COOH BLM-NH-ICFR lb) R-COX BLM-NH-fi-R lc) R-COOR BLM-NH f) R ld) R-CON BLM-NH-fi-R R-CO\ le) /O BLM--NHCR R-CO Ir) R C0-N l BLMNHfiR RCO\ lg) /0 BLMNHfiR RO-CO 0 CO lh) (CH o BLMNH-C-(NH,).CO0H

I Ii) CHCO /O BLMNH-C-CH,-CH--COOH CI-fi-CO CH =CCO 1 o BLMNHCCH,CCOOH CH,CO

CHC0 Ik) /O BLMNHCCH=CH--COOH CH-CO R'C-C0 n) BLM-NH, o BLMNHC-CH COOH H-co C0 HOOC R O R rm -co* BLMNH-CO- In the above-mentioned formulas, BLM represents the residue of the bleomycins exclusive of the side chain terminal amino group; R, R R, R", R R, R and n are as defined above; R is a lower alkyl or substituted or unsubstituted phenyl.

Of these modes of reaction, la) is a method for the direct N-acylation of the bleomycins with a carboxylic Propionic acid azide Butyric acid azide Acetic anhydride Propionic anhydride Butyric anhydride Octanoic anhydride Lauric anhydride Crotonic anhydride Phenylacetic anhyd ride acid in the presence of dehydrating agent such as a carbodiimide. The modes lb, lc, Id, le, lfand lg are meth- Phenoxyacetic anhydride ods for the N-acylation of the bleomycins with an acid halide, a carboxylic acid ester, a carboxylic acid azide, a carboxylic acid anhydride, an acylimidazole and a 25 Benzoic anh ydridc mixed acid anhydride obtained by a reaction of an alkoxyhalogenoformate with a carboxylic acid, respecp-Nitrobenzoic anhydride tively. The modes lh, li, lj, Ik, II and Im are methods for the N-acylation of the bleomycins with an intramolecular anhydride of a dibasic acid.

The representative compounds which may be used as a reagent in the present invention are as follows:

p-Nitrobenzoyl chloride Nicotinic anhydride Formic acid Acetic acid Propionic acid Butyric acid Valerie acid Caprylic acid Laurie acid Crotonic acid Benzoic acid Phenylacetic acid Z-Naphthoic acid p-Nitrobenzoic acid Acelyl chloride Propionyl chloride Chloroacetyl chloride Chloropropionyl chloride Bromoacetyl bromide Benzoyl chloride p-Nitrobenzoyl chloride Phenylacetyl chloride Phenoxyacetyl chloride Cinnamic anhydride Ethyl cinnamate Ethyl caprylate Benzoyl azide NO, @COOH CH;COCI C,H,COCl

ClCHgCOCl ClCH,CH,COCl BrCH,COBr

CDC]

COCl

OCH,COCl

lsonicotinic anhydride Furoic anhydride Thenoic anhydride Acetylimidazole Succinic anhydride Maleic anhydride Methylsuccinic anhydride ltaconic anhydride Citraconic anhydride Methyl chloride o cH,-co

No, co

CH CI Ethyl iseeyanate Methyl iodide Ethyl bromide liutyl hromldc Allyl bromide 'p-Nitrobenzyl chloride Benin bromide Metliylsull'onyl cnrmiat Ethylsull'onyl chloride Benzenesulfortyl chloride p-Toluenesulfonyl chloride p-Chlorobenzencsirll'onyl Chloride chloride t B-Nuphthalenesulfonyl :hioride Butyl isoeyanate v Phenyl isocyanate v Cyclohexyl isocyanate p-Methylphen'yl isocyanate mNaphthyl isocyanate B-Naphthyl isoeyanate Ethyl formirnidatehydrochloride Methyl acetimidate hydrochloride Methyl propioimidate Methyl valeroimidate hydrochloride Methyl pelargoniimidate v hydrochloride Methyl j-rhethylaminppropieimidate Methyl J-arninobutyroimidate hydrochloride Meth l J-dimethylathinoprnpioirnidate hydrochloride Methyl henziniidzile hydrochloride Methyl p-methoxyhenlimidate hydrochloride Methyl p-toluimidate hydrochloride Methyl m-nitrohenzimidate hydrochloride Methyl p-toluirnidate I hydrochloride Methyl p-bmmophenylacetimidate hydrochloride Methyl rh-ehlorophenylaeetimidate hydrochloride Methyl o-chlorophenylacctimidate hydrochloride -CH,IC;-OCH,.HCI

Methyl p-chlbrophenyl acetimidate hydrochloride Acrylonitrile CH,==CH CN The 'process of the present invention is carried out in a solvent selected from the group consisting of water. alcohols such as methanol and ethanol, ketones such as acetone, and a mixture thereof. The solvent is suitably selected in consideration of the-reagent used.

When lkyl halides such as'ethyl iodide, aeyl halides.

jalkyistllfcnyl halides are usedas a reagent, the reactiori velocity is fast and the use ofsueh'compounds in excesscauses .notgonly the desired 'monpsubstitution of' the side chain terminal'amino. group ofbleomycins but also".-.. the substitution of the' intranuciear amino group and' the (ii-substitution of the side-chain terminal amino group. Therefore, it is usually preferableto' use these compounds in-an amount of l to l.5 mols per mol of f bleomycinsQ-However, less reactive reagents'whieh do" not cause side reactions may be used in an amount as much as about it) mols permol of bleomycins. The reaction involved in the present invention is considered to be a nucleophilic substitution reaction of the terminal amino group of blcorriycins' and is preferably effeeted in an alkaline medium. For this purpose pyridine. sodium bicarbonate. triethylamine, etc. may be used. When imidate reagents are used, it is preferable to carry out the reaction at pH of 8.5 to.9.5 by the addition of the most strongly basic triethylarnine. However, the reaction for a long period of time at pH of, 10 or more should be avoided in viewofthe stability of bloc-- mycins. i

The reaction temperature may be generally 10 to 25C. ll'a reagent used is less reactive, however, the reaction is preferably carried out at. about 40CFor example, sulfonylation with various sulfonyl compounds ll l1 lcmpcmlul'c is completed in about 1 l 4 are in the form of blue powder as the starting bleomyhours. cins. They have.generally ultraviolet and infrared ab- For the separation of the dcs d P t lhc FCHC- sorption spectra similar to those ol'the starting materi- -.1i n liquid is neutralized to pH of? to 5 and.if necesals although the ultraviolet absorption spectra of the 581'). is COflCcfllfalcd fl fi 96 p Thc 5 derivatives containing special chromophores such as treated reaction liquid is then passed througha column.

packed with CM-Sephadex (-25 (tradename for a dry I CH ;/5'\ insoluble powder composed of' microscopic beads i f a which are synthetic organic compounds'eontaining ear; f

:hoxy methyl gr oup derived l',rom -the-polysaccharidcj d t -...dextran; manufactured and sold by Pharmacia Fine :1 v p Chemicals. l,nc.). The reaction p roductonce adsorhcd, V z 2 ;;s

- on the adsorbent is eluted with an aqueous salt solution such as ammonium chloride solution. The eluate frac-" I tion containing the desired product is passed through a are different from those ofthe starting materials.

column packed with active carbon or Amberlite CG-50 1 The products obtainedby the process of thepr esent i i exchange-resin manufactured invention were confirmed toibe the derivatives.

and sold by Rohmand Haas C0.). Theproduct once wherein their terminal amino group had been substiadsorbed on. the'absorbent is eluted with acetone-0.02 mi'a; by the Rt'value in thinlayer chromatography, the

N M'dtDChlO acid. l y v or methano -10 fact that they were differentin migration distance in 0.02 N hydrochlo aCid U l y C'OHCQIT- electrophoresis from the starting materials.- the 'fact trated and dried to obtain the desalted N-s'ubs'tituted v v ihat h w r llyn gati c in i h d i e i n derivative of a bleomyein,

Particularly. high mole'cuian substituted derivatives I trogenhad 'becn found.by thevan Slyke method.

such as 3-(N-lfiu ynfl p py fl y According to the process of thepresent invention sparingly soluble in water. Th ef r n s ethe mono'substituted derivatives are obtained as a V the reaction liquid shall be purified by ehromatograp l'ty-- main product anda small amount of the di-substituted and the fact that a decrease in the content of amino niusing a gel filtration for organic solvents such as Sephaderivatives are alsoproduced' depending on the reac-.

dcx Ll-i-ZO (t-radehame for dextran derivativeusing fortion conditions.

gelfiitrant in organic solvent, manufactured by Phar- The properties ofthe N-substituted derivative hydro- --rnacia Fine Chemicals Inc.), the desalting procedure, chlorides of bleomy'cins obtained according to the probeing unnecessary. cess of the present invention aresummarized in the fol- The formula, obtained amino-substituted derivative lowingTablc l.'

' Tablel j I Structure of terminal chain ho Reaent Name of derivative aminobleomycin' CaEhCO o absorption TLe R (Starting V HVE Ninhydrin meg Potency bleomycin) -mobility reaction per mg can '460 .1" 0

10,37 900 p.72 p 292 (0,001 240. 0.86 i530 292 "j(0'.62),- 4 so 24s p.88 9600 infrared absorption wave numher(cm' Ultraviolet '2" 4. 

1. A NOVEL N-SUBSTITUTED BLEMUCIN DERIVATIVE HAVING THE FOLLOWING STRUCTURE:
 2. The novel N-substituted derivative of claim 1 wherein said metal chelate is a copper chelate.
 3. A novel N-substituted bleomycin derivative as claimed in claim 1, wherein P represents
 4. A novel N-substituted bleomycin derivative as claimed in claim 1, wherein P represents -NH-(CH2)3-NH-(CH2)4-NH- and Q represents -COR (where R is a C1-C11 alkyl group, a nicotinyl group or an isonicotinyl group), -CO(CH2)n -COOH (where n1 is an integer of 2 to 7) or
 5. A novel N-substituted bleomycin compound having the following structure:
 6. An N-substituted bleomycin as claimed in claim 5, wherein P represents -NH-(CH2)3-NH-(CH2)4, -NH-,
 7. An N-substituted bleomycin as claimed in claim 5, wherein P represents -NH-(CH2)3-NH-(CH2)4-NH- and Q represents -COR (where R is a C1-C11 alkyl group, a nicotinyl group or an iso-nicotinyl group), -CO(CH2)n1-COOH (where n1 is an integer of 2 to 7) or
 8. The N-substituted bleomycin compound of claim 5 wherein A is a chlorine atom or 1/2 sulphate radical.
 9. The N-substituted bleomycin compound of claim 8 wherein A is a chlorine atom.
 10. A copper chelate of the compound of claim
 5. 11. A copper chelate of the compound of claim
 8. 12. A copper chelate of the compound of cliam
 9. 13. A novel N-substituted bleomycin derivative comprising an aminopropylaminobleomycin selected from the group consisting of 3-(N-4-Acetylaminobutyl)aminopropylaminobleomycin, 3-(N-4-Succinylaminobutyl)aminopropylaminobleomycin, 3-(N-4-Nicotinoylaminobutyl)aminopropylaminobleomycin, 3-(N-4-Isonicotinylaminobutyl)aminopropylaminobleomycin, 3-(N-4-Phthalylaminobutyl)aminopropylaminobleomycin, 3-(N-p-Chlorophenylacetimidoylaminobutyl)aminopropylaminobleomycin, 3-(N-Methyl-N-3-succinyl-aminopropyl)aminopropylaminobleomycin, 3-(N-Methyl-N-3-p -chlorophenylacetimidoylaminopropyl)aminopropylaminobleomycin, 3-(N-6-Succinylaminohexyl)aminopropylaminobleomycin, and 3-(N-6-p-Chlorophenylacetimidoylaminohexyl)aminopropylaminobleomycin.
 14. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-4-Acetylaminobutyl)aminopropylaminobleomycin.
 15. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-4-Succinylaminobutyl)aminopropylaminobleomycin.
 16. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-4-Nicotinoylaminobutyl)aminopropylaminobleomycin.
 17. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-4-Isonicotinoylaminobutyl)aminopropylaminobleomycin.
 18. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-4-Phthalylaminobutyl)aminopropylaminobleomycin.
 19. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-p-Chlorophenylacetiimidoylaminobutyl)aminopropylaminobleomycin.
 20. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-Methyl-N-3-succinylaminopropyl)aminopropylaminobleomycin.
 21. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-Methyl-N-3-p-chlorophenylacetiimidoylaminopropyl) -aminopropylaminobleomycin.
 22. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-6-Succinylaminohexyl)aminopropylaminobleomycin.
 23. The novel N-substituted bleomycin derivative of claim 13 wherein said aminopropylaminobleomycin is 3-(N-6-p-Chlorophenylacetiimidoylaminohexyl)aminopropylaminobleomycin.
 24. A process for producing N-substituted derivatives of bleomycins having the following structure in their copper-free form. 